The goal of this project is to develop improved methods for the synthesis, isolation anddetection of DNA/RNA based on the development of photocleavable oligonucleotide tags (PC-oligotags). PC-oligotags can be introduced into DNA/RNA chen~iicalfy during its solid-support synthesis by means of PC-phosphoramidites or enzymaticaffy using PC-nucleotides. The PC-oligonucleotides which result can be usefbl in a wide variety of applications in molecular biology including PCR-based site-directed mutagenesis, and DNA/RNA hybridization. In initial work, we have demonstrated that oligonucleotides can be 5'-labeled during their automated synthesis with PC-biotin phosphoramidite; selectively isolated from the crude oligonucleotide mixture by incubation with immobilized streptavidin and rapidly released by fight in a phosphorylated form for subsequent use in recombinant DNA methods. During Phase 1, we plan to synthesize and evaluate several promising PC-phosphoramidite and PC-nucleotide compounds. Improved methodology in molecular biology based on PC-oligotags will be evaluated including purification of full-length sequences from both synthetically produced DNA/RNA and PCR-based site-directed mutagenesis. During Phase II, PC-oligotags will be used for the automation of the synthesis, isolation and detection of oligonucleotides and will also be applied to specific problems in genetic and developmental biology.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
3P41RR010888-05S1
Application #
6478958
Study Section
Project Start
2000-07-01
Project End
2002-06-30
Budget Start
Budget End
Support Year
5
Fiscal Year
2001
Total Cost
$53,566
Indirect Cost
Name
Boston University
Department
Type
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
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